1. Field of the Invention
This invention relates to polynucleotides and more particularly to polynucleotides useful as primers in synthesizing deoxyribonucleic acids.
2. Description of the Prior Art
Highly purified interferom is only available for clinical trials in extremely small quantities. To permit the production of larger quantities of interferon and thus meet this need, human interferon is now synthesised in bacteria using genetic engineering methods (see S. Nagata, H. Taira, A. Hall, L. Johnsrud, M. Streuli, J. Escod i, W. Boll, K. Cantell and C. Weissmann in Nature 284, 316 (1980)).
For this, the gene which codes the protein sequence of the human leucocyte interferon which is to be produced has to be transferred into a bacterium in a suitable way so as to permit expression of the gene and hence synthesis of the human interferon in the bacterium. The procedure used is to integrate the corresponding deoxyribonucleic acid sequence in a self-replicating vector (preferably a bacterial plasmid or a bacterial virus) and then to introduce the genetic material modified in this way into the bacterial cell. To enable expression of this foreign gene to occur, the deoxyribonucleic acid sequence must be incorporated in the vector in the correct orientation and in the correct reading frame.
The nucleic acid suitable for this gene manipulation is appropriately selected on the messenger ribonucleic acid level (mRNA level), i.e. in order to recover the required DNA, the procedure is to synthesise the DNA which is complementary to the mRNA by means of an enzyme, known as reverse transcriptase. To initiate this enzymatic synthesis, a so-called primer is required, a fragment of DNA the base sequence of which is complementary to a portion of the mRNA in question and which, after being added to the mRNA, becomes bound to this complementary portion in the form of a hybrid double strand.
If the mRNA in question has portions containing poly-A, oligo-dT would be suitable as the primer. However, since the "correct" mRNA is not present in pure form but occurs in admixture with other mRNA sequences, this would initiate the transcription of all the mRNA sequences containing poly-A, and this would result in a complex mixture of DNA fragments which would not be suitable for the purpose specified above.
If, on the other hand, a deoxyoligonucleotide fragment which is complementary to a specific base sequence of the "correct" mRNA is used as the primer, there is a greater probability that only this one desirable transcription will occur, even in the presence of various other mRNAs, and hence the desired complementary DNA will be synthesised in a substantially pure state in the reverse transcriptase reaction.
The following application is based on the finding (see T. Taniguchi, N. Mantei, M. Schwarzstein, S. Nagata, M. Muramatsu and C. Weissmann in Nature 285, 547 (1980)), that all the interferon genes known at present have a common sequence consisting of thirteen nucleotides, which codes the amino acids nos. 47-50.
A sequence of thirteen nucleotides can be bound in stable fashion, in the form of a hybrid double strand (see M. Mevarech, B. E. Noyes and K. L. Agarwal in J. Biol. Chem. 254, 7472 (1979)) to all mRNA molecules which contain a sequence complementary thereto, but not to the mRNA molecules which occur predominantly in the present mixture and which do not contain this specific sequence. Therefore, if a specific DNA sequence of this kind is used as primer in the reverse transcriptase reaction, this results in a selection of the interferon-specific species of mRNA in the mixture of different nRNAs, and the probability of thus obtaining interferon-specific complementary DNA which is free from noninterferon-specific DNA is increased considerably.
Hence a need has continued to exist for a suitable primer for synthesis of DNA coding for interferon on an interferon mRNA matrix.